RESEARCH Open Access

Protein tyrosine phosphatase PTPN1modulates cell growth and associates withpoor outcome in human neuroblastomaCaroline E. Nunes-Xavier1,2*, Olaia Aurtenetxe1, Laura Zaldumbide3, Ricardo López-Almaraz4, Asier Erramuzpe5,Jesús M. Cortés5,6, José I. López1,3 and Rafael Pulido1,6,7*

Abstract

Background: Protein tyrosine phosphatases (PTPs) regulate neuronal differentiation and survival, but theirexpression patterns and functions in human neuroblastoma (NB) are scarcely known. Here, we have investigatedthe function and expression of the non-receptor PTPN1 on human NB cell lines and human NB tumor samples.

Material/methods: NB tumor samples from 44 patients were analysed by immunohistochemistry using specificantibodies against PTPN1, PTPRH, PTPRZ1, and PTEN. PTPN1 knock-down, cell proliferation and tyrosine phosphorylationanalyses, and RT-qPCR mRNA expression was assessed on SH-SY5Y, SMS-KCNR, and IMR-32 human NB cell lines.

Results: Knock-down of PTPN1 in SH-SY5Y NB cells resulted in increased tyrosine phosphorylation and cell proliferation.Retinoic acid-mediated differentiation of NB cell lines did not affect PTPN1 mRNA expression, as compared with otherPTPs. Importantly, PTPN1 displayed high expression on NB tumors in association with metastasis and poor prognosis.

Conclusions: Our results identify PTPN1 as a candidate regulator of NB cell growth and a potential NB prognosticbiomarker.

Keywords: Neuroblastoma, Neuroblastoma differentiation, Metastatic neuroblastoma, Protein tyrosine phosphatases,PTPN1

BackgroundNeuroblastoma (NB) constitutes a heterogeneous malig-nancy arising from sympathetic neural precursor cells fromthe neural crest. NB is the most common extracranialtumor in children, accounting in its high-risk forms forabout 15% of childhood cancer mortality. The majority ofNB tumors develop in the adrenal medulla, and, with lessfrequency, in the paraspinal ganglia from neck, chest, abdo-men, and pelvis [1–4]. Unbalanced neuronal differentiationseems to be an important aspect in NB initiation and pro-gression, with many primary and metastatic NB tumorsshowing spontaneous regression [5, 6]. High-risk NB donot show regression and are currently treated with a com-bination of chemo- and radio-therapy protocols, autologous

stem cell transplant, and surgery, followed by maintenancetherapies based on immuno-therapy and retinoic acid (RA)cell differentiating agents. However, high-risk NB are highlymalignant and frequently relapse after these combinationtherapies, rendering a survival lower than 50% of treatedchildren [7, 8]. Improvements in the definition of therapyresponse biomarkers and in the implementation of more ef-fective targeted therapies are current necessities in themanagement of high-risk NB patients [9–12].Tyrosine phosphorylation, executed by protein tyro-

sine kinases (PTKs), is one of the landmark protein post-translational modifications in human cancer [13]. Thereceptor tyrosine kinase (RTK) ALK is mutated in NBtumors and in the germline of patients with familial NB[14–16], and ALK plays a major role in the signal trans-duction mechanism driving NB development, being amajor potential target in NB targeted therapy [17, 18].ALK hyperactivation associates in NB with MYCN geneamplification, the major biomarker for high-risk NB, and

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: carnun@rr-research.no; rpulidomurillo@gmail.com;rafael.pulidomurillo@osakidetza.eus1Biomarkers in Cancer Unit, Biocruces Bizkaia Health Research Institute,Barakaldo, Bizkaia, SpainFull list of author information is available at the end of the article

Nunes-Xavier et al. Diagnostic Pathology (2019) 14:134 https://doi.org/10.1186/s13000-019-0919-9

potentiates MYCN oncogenic activity [19, 20]. Inaddition, the RTKs TrkA and TrkB are major signallingtransducers in the regulation of NB growth, differenti-ation, and apoptosis, whose expression has been corre-lated with NB prognosis and regression, and whosetherapeutic targeting is also being addressed in NB [21,22]. These examples illustrate that alterations in thetyrosine phosphorylation cellular status of NB cells arecrucial for NB development and progression. However,the regulated protein tyrosine dephosphorylation in NBhas been scarcely investigated.Protein tyrosine phosphatases (PTPs) are the direct

executers of dephosphorylation of specific tyrosineresidues on specific protein substrates, playing rele-vant roles in many physiologic and pathologic pro-cesses, including those related with the regulation ofcell differentiation, transformation, and growth [23–27]. Inhibition of PTPs by vanadium compounds en-hances RA-triggered differentiation of NB cells, sug-gesting an active role for these enzymes in NB cellproliferation and senescence [28]. Moreover, PTP in-hibition by vanadate or vanadium compounds courseswith induced cell death on NB cell lines, making PTPinhibition a suitable therapeutic option in NB [29].The non-receptor tyrosine phosphatase PTPN1 (alsoknown as PTP1B) constitutes the paradigm of PTPenzymes and a suitable drug target for cancer andmetabolic diseases [30–33], and PTPN1 protein ex-pression has been shown to correlate with metastasisand poor prognosis in several human cancers [34–37].In this study, analysing human NB tumor samplesand cell lines, we have found evidence for PTPN1 asa regulator of NB cell tyrosine phosphorylation andproliferation, and unveiled the association of PTPN1expression with poor NB patient outcome.

MethodsPatients, tissue specimens, and immunohistochemistryThe characteristics of the patients included in the studyhave been previously described [38] (Table 1). Histo-logical sections of tissue microarrays (TMAs) or routineparaffin blocks containing the tumor specimens wereused for immunohistochemistry (IHC). The antibodiesand dilutions used for IHC were: PTEN (1/50, 6H2.1,Merck Millipore), PTPRZ1 (1/50, Clone 12/RPTPb, BDBioscience), PTPRH (1/500, HPA042300, Sigma-Aldrich), and PTPN1 (1/20, AF1366, R&D). Immunos-tainings were performed in automated immunostainers(EnVision FLEX, DakoAutostainer Plus; Dako,Glostrup, Denmark) following routine methods. Theanalysis was done blind by an experienced pathologist(LZ) and performed using a Nikon Eclipse 80i micro-scope (Tokyo, Japan). The IHC evaluation consideredpositive (high) those cases with intense nuclear or

granular cytoplasmic staining positive cells, and nega-tive (low/no) those with weak or non-existent stainingpositive cells). Each examined core and routine paraffinblock contained a minimum of 200 tumor cells.

Cell lines and reagentsHuman NB cell lines SH-SY5Y (ALK F1174 L), SMS-KCNR (ALK R1275Q, MYCN amplified), and IMR-32(MYCN amplified) are from ATCC. Cells were grownat 37 °C in a humidified 5% CO2, 95% air incubator.SH-SY5Y and IMR-32 cells were grown in DMEM/F12 supplemented with 10% FBS, 2 mM L-glutamine,100 units/ml of penicillin, 0.1 mg/ml of streptomycin,and 1% non-essential amino acids. SMS-KCNR weregrown in DMEM supplemented with 10% FBS, 2 mML-glutamine, 100 units/ml of penicillin, and 0.1 mg/mlof streptomycin. The three cell lines differentiate

Table 1 Clinic-pathologic characteristics and MYCNamplification of study population

Patients (n = 44) % MYCN

amp No-amp

Gender (p = 0.604)(R = -0.090)

Male (21) 48 3 12

Female (23) 52 5 13

Age at diagnosis (months) (p = 0.023)(R = 0.396)

< 18 (29) 66 3 20

> 18 (15) 34 5 5

Risk (p = 0.003)(R = 0.516)

Intermediate, high (23) 52 8 10

Low (21) 48 8 0

Stage (p = 0.002)(R = 0.550)

Metastatic (12) 27 6 4

Non-metastatic (32) 73 2 21

Stage (p = 0.018)(R = 0.412)

III, IV (17) 39 6 7

non-III, non-IV (27) 61 2 18

Stage (p = 0.000)(R = 0.606)

IV (13) 30 6 3

non-IV (31) 70 2 22

Survival (p = 0.000)(R = 0.650)

Dead (10) 23 5 1

Alive (34) 77 3 24

Note that information on MYCN amplification is not available for all samplesSignificant p values (p < 0.05) are in bold

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upon RA treatment [39–41]. Cell differentiation wasinduced by adding 10 μM all-trans retinoic acid (RA)(Sigma) to the cultures, followed by 10 days incuba-tion (media was changed after 5 days).

mRNA isolation and RT-qPCRRT-qPCR was performed using RNA from SH-SY5Y,SMS-KCNR, and IMR-32 cells treated or not with RA,using the IllustraRNAspin mini purification kit (GEHealthcare Life Sciences). 1 μg of total RNA was re-verse transcribed using RevertAidTM reverse tran-scriptase, oligo (dT)18 primers, and RiboLock andRNase inhibitor (all from Fermentas). qPCR was per-formed as previously described [42] using validated pri-mer sets (Qiagen) specific for the classical PTPs andreference gene (hypoxanthine phosphoribosyltransfer-ase 1 [HPRT1]). All quantifications were normalized tothe HPRT1 reference gene data. Relative quantificationwas performed using the comparative ΔΔCt method.Significant up-regulation or down-regulation was con-sidered with a threshold of 2 or − 2 fold change (Log2scale), respectively. Non-significant up-regulation ordown-regulation was considered between 1 and 2 foldchange or between − 1 and − 2 fold change, respectively.Those classical PTPs displaying coordinated significantincreases in the three cell lines were considered asPTPs with significant up-regulation, whereas thosePTPs displaying coordinated significant changes in twocell lines and non-significant changes in one cell linewere considered as PTPs with significant up-regulationor down-regulation trends.

PTPN1 knock-down, MTS cell proliferation andimmunoblot assaysPTPN1 knock-down was performed by transfection ofspecific siRNAs using Lipofectamine 3000 (ThermoFisher) or PepMute (SignaGen) reagents followingmanufacturer’s protocol. PTPN1 siRNAs (siPTPN1 #1,SI00043827; siPTPN1 #2, SI00043806) were from Qia-gen, and siNS (non-specific) and siGAPDH siRNAswere from Ambion. PTPN1 knock-down was verified72 h post-transfection at mRNA level by isolation ofRNA and RT-qPCR as described above. Cell prolifera-tion was analyzed 72 h post-transfection using CellTi-ter 96® AQueous One Solution Cell Proliferation Assay(MTS) (Promega) following manufacturer’s protocol.For immunoblot analysis, SH-SY5Y cells were incu-bated in the presence of epidermal growth factor(EGF; 50 ng/ml) for 5 min, and whole cell protein ex-tracts were prepared by cell lysis in ice-cold M-PER™lysis buffer (ThermoFisher Scientific) supplementedwith PhosSTOP phosphatase inhibitor and cOmpleteprotease inhibitor cocktails (Roche, Switzerland),followed by centrifugation at 15200 g for 10 min and

collection of the supernatant. Proteins (50 μg) were re-solved in 10% SDS-PAGE under reducing conditionsand transferred to PVDF membranes (Merck Milli-pore). Immunoblotting was performed using anti-phosphotyrosine 4G10 (1/1000, 05–321, Merck Milli-pore) and anti-GAPDH (1/1000, sc-32,233, Santa CruzBiotechnology) antibodies, followed by IRDye second-ary antibody (LI-COR) and visualization by Odyssey®CLx Imaging System. Electrophoretic bands werequantified using ImageJ software.

Statistical analysis of immunohistochemistry dataStatistical analysis was performed using IBM SPSSStatistics package, and we performed crosstabs Pear-son Chi square analysis, using 2-sided asymptotic Sig-nificance to calculate p-values, and interval by intervalsymmetric measures for Pearson’s R value.

ResultsPTPN1 knock-down and growth of NB cellsTo investigate the impact of the expression of PTPN1 inthe modulation of growth of human NB cells, SH-SY5Ycells were transiently transfected with specific siRNAs tar-geting PTPN1 for down-regulation (Fig. 1a), and phospho-tyrosine content and MTS cell proliferation assays wereperformed 72 h after transfection. As shown, EGF short-term stimulation triggered tyrosine phosphorylation inSH-SY5Y cells, which was enhanced upon PTPN1 silen-cing, when compared to non-specific silencing conditions(Fig. 1b). Accordingly, siRNA down-regulation of PTPN1expression resulted in increased cell proliferation (Fig. 1c).These results suggest a linkage between tyrosine phos-phorylation status and proliferation of SH-SY5Y cells andillustrate the involvement of PTPN1 in the regulation ofcell signaling and growth in NB.

PTPN1 expression in human NB cells and NB tumorsamplesThe expression patterns of PTPN1 in human NB cellsand NB tumor samples was investigated, in compari-son with other PTPs, including PTPRH, PTPRZ1, andPTEN. The mRNA expression of these PTPs in thehuman adrenal gland (the major neuroendocrine tis-sue source of NB) and in the SH-SY5Y human NBcell line (the more studied human NB cell line) isshown in Fig. 2a and b, respectively. PTPN1 mRNAis abundantly detected from both of these sources,whereas other PTP mRNAs, such as PTPRZ1, are notdetected. We also explored the potential role of thesePTPs in NB differentiation, performing a quantitativeRT-PCR (RT-qPCR) analysis of their mRNA expres-sion on human NB cell lines differentiated in thepresence of all-trans RA. These included SH-SY5Y,SMS-KCNR, and IMR-32 cell lines, which show

Nunes-Xavier et al. Diagnostic Pathology (2019) 14:134 Page 3 of 8

different genetic backgrounds in terms of ALK muta-tional status and MYCN amplification. The cell lineswere grown in the absence or in the presence of RAfor 10 days, and mRNA was isolated and subjected toRT-qPCR using specific PTP oligonucleotide primers[42]. Figure 2c shows the relative changes of PTPmRNA expression from RA-treated cells in compari-son with untreated cells. PTPN1 expression was notaltered upon RA cell treatment, whereas the expres-sion of PTPRH and PTPRZ1 was significantly up-regulated, and that of PTEN slightly up-regulated, inthe three NB cell lines analysed.Next, the expression on human NB tumor samples of

PTPN1, PTPRH, PTPRZ1, and PTEN was analyzed by

IHC using specific antibodies. A summary of our IHCresults is shown in Table 2, and illustrative NB immuno-stained sections are shown in Fig. 3. PTPRH andPTPRZ1 were expressed in a large number of NB tumorsamples, mostly displaying a cytoplasmic granular stain-ing pattern. PTPRZ1 was expressed with high intensityin most of the tumor samples, suggesting that it couldbe a good marker in NB. High staining of PTPRH asso-ciated with low risk and non-IV stage. PTEN tumor sup-pressor was expressed in the majority of NB tumorsamples and no clinical correlations were found, inagreement with previous reports [43], indicating that thelack of PTEN is not a common driving event in NB [44].PTPN1 was also positive or weakly positive in most of

Fig. 1 Tyrosine (Tyr) phosphorylation and proliferation of SH-SY5Y cells upon PTPN1 siRNA knock-down. Cells were transfected withcontrol (siNS, non-specific) siRNAs or with two different siRNAs targeting PTPN1 (siPTPN1 #1, siPTPN1 #2), and assays were performed 72 hafter transfection. a The silencing efficiency of the distinct siRNAs is shown, as monitored by RT-qPCR using specific primers. bPhosphotyrosine cell content was analysed by immunoblot with anti-phosphotyrosine 4G10 mAb (pY). GAPDH immunoblot was run asloading control. In the upper panel, a representative experiment is shown. In the lower panel, the quantification of phosphotyrosinecontent from each condition is shown. Results are shown as phosphotyrosine content normalized to GAPDH content (pY/GAPDH). c MTScell proliferation; AU, arbitrary units. Data are shown as the mean + SD from two technical replicates from a representative experiment (aand c) or as the mean + SD from two independent experiments (b)

Nunes-Xavier et al. Diagnostic Pathology (2019) 14:134 Page 4 of 8

the cases, with a cytoplasmic granular immunostain-ing. Importantly, high PTPN1 expression correlatedwith poor outcome parameters, including intermedi-ate/high risk, metastasis and stages III/IV (Table 2).Further (Chi square) analysis revealed more significantcorrelations with low risk (p = 0.001, R = -0.501), non-metastatic (p = 0.042, R = -0.306), non-IV stage (p =0.003, R = -0.448), and non-III/non-IV stage (p = 0.005,R = -0.420) for samples displaying high PTPRH andlow PTPN1 expression. In conclusion, our findingssuggest a differential involvement of PTPN1 andPTPRH in malignant NB progression, and argue forPTPN1 high expression as a potential surrogatemarker for poor outcome NB.

DiscussionThe efficacy of high-risk NB novel targeted therapiesrelies on a precise patient stratification based on novel

genetic, epigenetic, or protein biomarkers [10, 45, 46].In our study, we have found association of PTPN1high expression with NB poor outcome, as indicatedby PTPN1 enrichment in metastatic disease, and insamples from high stage and intermediate/high-riskNB patients. Interestingly, PTPRH expression dis-played inverse clinical correlations than PTPN1 ex-pression, which were increased when PTPRH high andPTPN1 low cases were clustered. This suggestsPTPRH+/PTPN1- immunostaining of NB tumors as agood molecular predictor of low risk NB. Leptin or in-sulin treatment induced PTPN1 mRNA expression inSH-SY5Y cells [47]. However, PTPN1 mRNA expres-sion did not display significant changes upon RA-induced differentiation of human NB cells (this re-port). On the other hand, high expression of PTPRHand PTPRZ1, which were coordinately up-regulated byRA in NB cell lines, was detected in our set of NB

Fig. 2 a mRNA expression of PTPs in Adrenal gland. Data from GTEx (Genotype-Tissue Expression) data sets. b mRNA expression of PTPs in SH-SY5Y cells. Data from HPA (Human Protein Atlas). Data in a. and b. are reported as transcription expression NX values (Normalized eXpressiondata, and is based on RNA-seqdata), according to https://www.proteinatlas.org/. c mRNA expression analysis of PTPs from human NB cell linestreated with RA. Cell lines were kept untreated or were treated for 10 days with RA, mRNA was extracted and RT-qPCR was performed usingspecific PTP primers. Relative mRNA expression values are shown in Log2 as fold change + S.D. of treated cells versus untreated cells, from atleast two independent experiments. Mean fold change above 2 or below −2 was considered significant

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tumors. Interestingly, up-regulation of PTPRH hasalso been detected in breast cancer cell lines grownupon differentiation conditions [48]. How these PTPexpression patterns in NB cells could be related withthe differential expression and function of PTPs in NBtumors deserves further investigation. Noticeably,siRNA knock-down of PTPN1 resulted in increasedtyrosine phosphorylation and cell proliferation of SH-SY5Y cells, indicating that PTPN1 could be importantin regulating NB cell growth. This is in agreementwith the report from Ozek et al. showing that PTPN1inhibition increased the tyrosine phosphorylation anddownstream signaling of TrkB, as well as the neurite

outgrowth, in SH-SY5Y cells [49]. In addition, PTPN1has been proposed to dephosphorylate the intracellu-lar pools of ALK in mouse fibroblasts [50]. Together,our findings suggest that PTPN1 high expression inNB tumors might not be necessary for NB cell trans-formation, but rather could arise as a surrogatemarker of NB tumor evolution, and support the hy-pothesis that PTPN1 growth-modulatory activity inSH-SY5Y NB cells takes place upstream in tyrosinephosphorylation signaling pathways. Further work isgranted to understand the involvement of PTPN1 inNB cells growth and to validate PTPN1 as a novel NBprognostic biomarker.

Table 2 Immunostaining of PTPs from NB tissue sections

Patients (n = 44) % PTPN1 PTPRH PTPRZ1 PTEN

high low/no high low/no high low/no high low/no

Gender (p = 0.3179)(R = 0.155)

(p = 0.571)(R = 0.068)

(p = 0.832)(R = 0.032)

(p = 0.4475)(R = 0.115)

Male (21) 48 8 11 17 3 17 3 19 2

Female (23) 52 6 16 18 5 19 4 19 4

Age at diagnosis (months) (p = 0.691)(R = 0.62)

(p = 0.243)(R = -0.178)

(p = 0.525)(R = -0.097)

(p = 0.376)(R = -0.133)

< 18 (29) 66 9 19 25 4 25 4 26 3

> 18 (15) 34 8 5 10 4 11 3 12 3

MYCN (p = 0.2551)(R = 0.204)

(p = 0.1036)(R = 0.283)

(p = 0.3719)(R = -0.155)

(p = 0.9699)(R = -0.007)

Amplified (8) 18 4 3 5 3 6 0 7 1

Non-amplified (25) 57 8 16 22 3 22 3 22 3

Risk (p = 0.013)(R = 0.394)

(p = 0.023)(R = -0.348)

(p = 0.241)(R = -0.179)

(p = 0.448)(R = -0.115)

Intermediate, high(23) 52 10 11 15 7 17 5 19 4

Low (21) 48 3 17 20 1 2 19 19 2

Stage (p = 0.047)(R = 0.310)

(p = 0.079)(R = 0.268)

(p = 0.335)(R = 0.147)

(p = 0.530)(R = 0.095)

Metastatic (12) 27 6 4 7 4 9 3 11 1

Non-metastatic (32) 73 8 23 28 4 27 4 27 5

Stage (p = 0.008)(R = 0.414)

(p = 0.101)(R = .250)

(p = 0.735)(R = -0.052)

(p = 0.234)(R = 0.179)

III, IV (17) 39 9 6 11 5 13 3 16 1

non-III, non-IV (27) 61 5 21 24 3 23 4 22 5

Stage (p = 0.095)(R = 0.260)

(p = 0.001)(R = -0.502)

(p = 0.059)(R = -0.287)

(p = 0.827)(R = -0.033)

IV (13) 30 6 5 6 6 8 4 11 2

non-IV (31) 70 8 22 29 2 28 3 27 4

Survival (p = 0.824)(R = 0.159)

(p = 0.2016)(R = -0.195)

(p = 0.1191)(R = -0.238)

(p = 0.505)(R = -0.101)

Dead (10) 23 3 5 6 3 6 3 8 2

Alive (34) 77 11 22 29 5 30 4 30 4

PTP immunostaining was not obtained in some cases due to lack of tissue to perform the analysis or to non-informative immunostaining resultsSignificant p values (p < 0.05) are in bold

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ConclusionPTPN1 high expression in NB tumors associated withpatient poor outcome, and PTPN1 knock-down affectedthe tyrosine phosphorylation status and proliferation ofNB cells. This makes PTPN1 a suitable candidate for NBprognostic and intervention marker.

AbbreviationsIHC: Immunohistochemistry; NB: Neuroblastoma; PTK: Protein tyrosine kinase;PTP: Protein tyrosine phosphatase; RA: Retinoic acid; RTK: Receptor tyrosinekinase; TMA: Tissue microarray

AcknowledgementsWe thank Basque Country Biobank for help and support, and Arantza PerezDobaran (Department of Physiology, Universidad del País Vasco, UPV/EHU,Leioa, Bizkaia, Spain) for expert technical assistance.

Authors’ contributionsCENX, OA, LZ, RL, JIL, and RP conceived and designed the experimentalanalysis. CENX, OA, and LZ performed the experiments and collected data.CENX, OA, LZ, RLA, AE, JC, JIL, and RP contributed materials and analysistools, and interpreted data. CENX and RP wrote the paper. All authors readand approved the final manuscript.

FundingThis work was partially supported by grants: BIO13/CI/001/BC from BIOEF(EITB maratoia), Basque Country, Spain; SAF2013–48812-R and SAF2016–79847-R from Ministerio de Educación y Ciencia (to RP) and Ministerio deEconomía y Competitividad (Spain and Fondo Europeo de DesarrolloRegional) (to RP and JIL); and 239813 from The Research Council of Norway

(to CENX). AE was supported by a predoctoral grant from the BasqueGovernment (Programa de Formación de Personal Investigador no doctor,Departamento de Educación, Política Lingüística y Cultura del GobiernoVasco).

Availability of data and materialsAll data generated or analysed during this study are included in thispublished article.

Ethics approval and consent to participateAll procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional and/or nationalresearch committee (Ethical and Scientific Committees of the BasqueCountry Public Health System, Osakidetza, INC + CES BIOEF2015–03) and withthe 1964 Helsinki declaration and its later amendments or comparableethical standards. Written informed consent was obtained from allparticipants.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Biomarkers in Cancer Unit, Biocruces Bizkaia Health Research Institute,Barakaldo, Bizkaia, Spain. 2Department of Tumor Biology, Institute for CancerResearch, Oslo University Hospital Radiumhospitalet, P.O. Box 4950 Nydalen,N-0424 Oslo, Norway. 3Department of Pathology, Cruces University Hospital,University of the Basque Country (UPV/EHU), Barakaldo, Bizkaia, Spain.4Pediatric Oncology and Hematology, Cruces University Hospital, Barakaldo,Bizkaia, Spain. 5Quantitative Biomedicine Unit, Biocruces Bizkaia HealthResearch Institute, Barakaldo, Bizkaia, Spain. 6IKERBASQUE, Basque Foundationfor Science, Bilbao, Spain. 7Biocruces Bizkaia Health Research Institute,Hospital Universitario de Cruces, Plaza Cruces s/n, 48903 Barakaldo, Spain.

Received: 15 October 2019 Accepted: 6 December 2019

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